A fat-depleted adipose tissue and a device and method for preparing the same

ABSTRACT

The present invention provides a device configured to mechanically remove a fat phase from at least one adipose tissue, the device comprising a first pressing element and a second pressing element. Furthermore, there is provided a method for preparing a preparation comprising a fat-depleted adipose tissue, the method comprising: mechanically removing a fat phase from at least one adipose tissue. In addition, there is provided a preparation comprising a fat-depleted adipose tissue, wherein the fat-depleted adipose tissue is prepared by mechanically removing a fat phase from at least one adipose tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional patentapplication No. 62/156,935, filed May 5, 2015.

FIELD OF THE INVENTION

The present invention relates to tissue regeneration. More particularly,the present invention relates to the provision of preparations for boneand soft tissue regeneration.

BACKGROUND OF THE INVENTION

A bone is a rigid organ that constitutes part of the vertebral skeleton.Bones serve multiple functions, including support and protection oforgans of the body, production of red blood cells and lymphocytes,storage of minerals and enablement of mobility of the body. Bones arelightweight yet strong and hard, come in a variety of shapes and sizesand have a complex internal and external structure. There are two typesof bone tissue in a bone: cortical and cancellous. The cortical bonetissue forms the cortex, or outer shell of the bone. It is a compact,dense, hard, strong and stiff tissue, filled with a mineralized matrixmade of an organic component, mainly collagen, and an inorganiccomponent of bone mineral made up of various salts, mainly salts ofcalcium and phosphate. In the mineralized matrix there are tiny spaces,termed lacunae, which contain bone cells. The cancellous tissue is foundin the marrow space (medullary cavity) of the bone, and has asponge-like appearance with numerous large spaces. Other types of tissuefound in bones include marrow—where red blood cells and lymphocytes areproduced, endosteum—the tissue lining the medullary cavity of the cone,periosteum—a thin connective tissue that covers the surface of the bone,nerves, blood vessels and cartilage.

There are four types of bone cells in a bone: osteoblasts, osteocytes,lining cells and osteoclasts.

The osteoblasts are mononucleate small cells that are responsible forthe formation of bone tissue, and thus have an important role in boneformation, remodeling and repair. During bone formation, osteoblastsfirst secret a unique collagen-rich extracellular matrix, which serve asa framework on which mineralization of the bone tissue is taking placeby deposition of calcium phosphate, which is then hardened by hydroxideand bicarbonate ions. During bone formation, some osteoblasts aretrapped in the hardened bone tissue, while others remain on top of thenew bone tissue.

Osteocytes are osteoblasts that were trapped in the newly formed bonetissue. They reside in tiny spaces in the bone tissue, termed lacunae.Osteocytes communicate with each other through long cellular extensions,sense mechanical stress in the bone, and send signals to the osteoblastsfor bone remodeling as a result of mechanical stress.

Lining cells are osteoblasts that remained on top of the new bonetissue, and used to protect the underlying bone tissue.

Osteoclasts are multinucleate large macrophage-like cells that areresponsible for the breakdown of bones. Osteoclasts resorb bones by acombination of localized acidification for removing the minerals andprotease secretion for breaking down the collagen matrix. Osteoclaststhat tunnel through the bone are usually followed close behind byosteoblasts, which form a new bone tissue. This remodeling process isvery important for bone health. Therefore, because of its importance inbone remodeling, the breakdown of bones is controlled by hormones, whichinstruct the osteoclasts when and where to break down bone tissue.

Bone formation takes place during fetal development of the skeletalsystem and wound healing. Two distinct processes are involved in boneformation: endochondral ossification and intramembranous ossification.During the endochondral ossification cartilage is first formed bymesenchymal progenitor cells. Then, cartilage cells, known aschondrocytes, undergo hypertrophy and the extracellular matrixmineralizes. Then, blood vessels invade the newly formed tissue,bringing cells that degrade the existing cartilage matrix. Afterwardsbone tissue is formed by osteoprogenitor cells. Long bones are formed byendochondral ossification during fetal development. On the other hand,intramembranous ossification is a more direct process, in whichosteoprogenitor cells form bone tissue directly. Carnial bones areformed by intramembranous ossification during fetal development. Woundhealing in a bone may proceed with either process, depending on localenvironmental factors, including how much the ends of the bone can moverelative to each other. For example, in cases when the ends of the bonecan significantly move relative to each other, endochondral ossificationis favored.

Osteoprogenitor cells have the ability to differentiate into osteoblastsor chondrocytes, depending on the signaling molecules they are exposedto, giving rise to either bone or cartilage, respectively. These cellsreside in the periosteum and the marrow of a bone. The osteoprogenitorcells are progenitor cells that arise from mesenchymal stem cells.

Bone regeneration is a complex physiological process of bone formationoccurring during normal fracture healing and is involved in continuousbone remodeling. However, there are complex clinical conditions in whichbone regeneration is required in a large extent, for example duringskeletal reconstruction of large bone defects created by major trauma,infection, tumor resection or skeletal abnormalities; or in cases inwhich the regenerative process is compromised, for example vascularnecrosis, atrophic nonunion and osteoporosis.

In some cases, such as dental reconstruction and nonunion fracture,there may be enough progenitor cells in the damaged area that may bestimulated to induce local bone formation. However, in other cases, forexample a compromised site, such as where a tumor was removed and thelocal tissue was irradiated, there may not be enough local progenitorcells to induce local bone formation. Also an alternative approach, suchas releasing growth factors in the damaged site in order to induce theformation of progenitor cells, in a site where a tumor was removed, maynot be a good idea.

Another approach is soft tissue regeneration, which includes for examplegum tissue grafts such as connective tissue grafts, free gingival graftsand pedicle grafts. There are cases in which tissue-stimulating proteinsare used to encourage the natural ability of the body to support growthof the tissue.

An example of a soft tissue regeneration procedure is gingivalreconstruction. The gingiva surrounding a tooth has a 2-3 mm band ofbright pink, very strong attached mucosa, then a darker, larger area ofunattached mucosa that fold into the cheeks. When replacing a tooth withan implant, a band of strong, attached gingiva is needed to keep theimplant healthy in the long-term. This is especially important withimplants because the blood supply is more precarious in the gingivasurrounding an implant, and is theoretically more susceptible to injurybecause of a longer attachment to the implant than on a tooth (a longerbiologic width). When an adequate band of attached tissue is absent, itcan be recreated with a soft tissue graft.

According to Rozalia Dimitriou et al (2011): “Currently, there is aplethora of different strategies to augment the impaired or‘insufficient’ bone-regeneration process, including the ‘gold standard’autologous bone graft, free fibula vascularized graft, allograftimplantation, and use of growth factors, osteoconductive scaffolds,osteoprogenitor cells and distraction osteogenesis. Improved ‘local’strategies in terms of tissue engineering and gene therapy, or even‘systemic’ enhancement of bone repair, are under intense investigationin an effort to overcome the limitations of the current methods, toproduce bone-graft substitutes with biomechanical properties that are asidentical to normal bone as possible, to accelerate the overallregeneration process, or even to address systemic conditions, such asskeletal disorders and osteoporosis.” (Rozalia Dimitriou, Elena Jones,Dennis McGonagle and Peter V. Giannoudis. BMC Medicine, 2011, 9:66doi:10.1186/1741-7015-9-66).

In general, current clinical treatments for critical-sized bone defectsare problematic and often yield poor healing due to the complicatedanatomy and physiology of bone tissue, as well as the limitations ofmedical technology.

Bone regeneration procedures include procedures such as correctiondefects in bones (caused by traumas, diseases etc.); adding missing boneparts; spinal fusion surgery; and maxillary sinus lifting procedure. Inaddition, the bone regeneration procedure may include the usage ofcages.

General applications of bone regeneration include: healing of nonunionfractures; craniofacial reconstruction; healing of segmental defects dueto tumor removal or trauma; augmentation of bone around hip implantsrevision; and spinal fusion.

Applications of bone regeneration in dental procedures include: buildingup bone tissue around implants placed in tooth sockets after toothextraction; socket preservation for future implantation of false teethor prosthetics; filling of bone defects after removal of the root of atooth, cystectomy or removal of impacted teeth; and repairing bonedefects after reopening of a wound.

One example of bone regeneration procedure is a sinus lifting. A sinuslift is a bone grafting procedure typically done when the bone in apatient's upper jaw is too thin to securely hold dental implants. Theprocedure involves significant trauma. Six to nine months after theprocedure, new bone is generated.

Bone tissue engineering offers a promising alternative strategy ofhealing severe bone injuries by utilizing the body's natural biologicalresponse to tissue damage in conjunction with engineering principles.Osteogenic cells, growth factors, and biomaterial scaffolds form thefoundation of the many bone tissue engineering strategies employed toachieve repair and restoration of damaged tissue. An ideal biomaterialscaffold will provide mechanical support to an injured site and alsodeliver growth factors and cells into a defect to encourage tissuegrowth. Additionally, this biomaterial should degrade in a controlledmanner without causing a significant inflammatory response.

An important element for bone regeneration is having a source that willprovide viable cells that can differentiate and proliferate intoosteogenic cells. For a long time, autologous bone graft has been usedand considered as the gold standard material for bone regeneration inorthopedic surgery. Autologous bone is usually harvested from theanterior and posterior iliac crests of the pelvis. It can also beharvested as vascularized bone graft containing an internal vascularnetwork in order to restore a significant bone defect, or tricorticalgraft for structural support. Although autologous bone graft is a safeand effective way to provide bone cells still it has multiplelimitations including donor site morbidity, limited cells quantity,requirement of a second surgical procedure with frequent consequences ofpain, and complications (Mina W. Morcos, Hadil Al-Jallad and ReggieHamdy, 2015, Review article—Comprehensive Review of Adipose Stem Cellsand Their Implication in Distraction Osteogenesis and Bone Regeneration,BioMed Research International, Volume 2015, Article ID 842975,http://dx.doi.org/10.1155/2015/842975, referred to hereinafter as Morcoset al, 2015).

An alternative method to harvest autologous bone graft is reamerirrigation aspiration system. Reamer irrigation aspiration is anintramedullary reaming system that provides continuous irrigation andaspiration during intramedullary reaming. It was originally designed todecrease the adverse effects of reaming long bone fractures bycollecting the reaming material which contains a significant number ofosteogenic cells. This provides a large volume of corticocancellous bonematerial that can be used as autologous bone graft. This is usuallyharvested from the femur [89]. This technique provides a large volume ofautologous bone graft that corresponds to the bone graft obtained fromboth the anterior and posterior iliac crest; however it still hassimilar limitations regarding the need for a second operation and thelimited quantity of cells that can be provided (Morcos et al, 2015).

Another method would be allograft bone that is available in differentpreparations. However, allograft bone lacks osteogenic capacity as itdoes not contain living bone cells; therefore it is not considered to bea good source for osteogenic cells. Moreover it carries the risk ofdisease transmission and immunogenic responses (Morcos et al, 2015).

Since both autograft and allograft have restrictions, the use ofmesenchymal stem cells has been considered.

Mesenchymal stem cells are multipotent stromal cells than candifferentiate and proliferate into a variety of cell types, including:chondrocytes (cartilage cells), myocytes (muscle cells), adipocytes (fatcells), and osteogenic cells under the appropriate molecular signals.Mesenchymal stem cells have been found in multiple tissues includingbone marrow and adipose tissues, and growth factors may be used toenhance cell proliferation and differentiation of mesenchymal stem cellsinto osteogenic lineage (Morcos et al, 2015).

Stem cells are undifferentiated progenitor cells that are capable ofboth self-renewal and multilineage differentiation. They are classifiedinto two categories, depending on their origin: the embryonic stem cellsand adult stem cells. Adult stem cells are derived from differentiatedpostnatal tissues and are believed to be intimately involved in tissueand organ regeneration and repair during injury and ageing. Adult stemcells are considered to be multipotent since they have a low degree ofplasticity (Morcos et al, 2015).

Mesenchymal stem cells are a type of adult stem cells, which wereinitially discovered in bone marrow, but afterwards were isolated andcharacterized from several adult and fetal tissues, including adiposetissue, dermis, periosteum, umbilical cord blood, placenta and amnioticfluid, and synovial fluid. Mesenchymal stem cells have significanttherapeutic potentials that can be applied to multiple disciplinesespecially where Mesenchymal stem cells show low immunogenicity.Mesenchymal stem cells can differentiate into osteoblasts,chondroblasts, and adipocytes (Morcos et al, 2015).

Adipose derived mesenchymal stem cells are able to differentiate intomultiple lineages, in particularly chondrocytes, osteocytes, andadipocytes, when the correct conditions are provided. The induction ofAdipose derived mesenchymal stem cells differentiation in vitro isachieved by culturing Adipose derived mesenchymal stem cells in specificmedia. Another practice to differentiate Adipose derived mesenchymalstem cells is the application of physical stimuli including mechanicalforces, magnetic, and electrical fields (Morcos et al, 2015).

Adipose derived mesenchymal stem cells are isolated from lipoaspirates.One gram of

adipose tissue yields approximately 3×10⁵-1×10⁶ mesenchymal stem cells,which is 500-fold greater than the number of mesenchymal stem cells inone gram of bone marrow. Adipose derived mesenchymal stem cells possessseveral advantages when compared to bone marrow mesenchymal stem cells.First, Adipose derived mesenchymal stem cells are readily available inlarge quantities, almost unlimited, and can be retrieved in high volumesof cellular population with less invasive methods such as liposuctionaspirates or subcutaneous adipose tissue fragments. Moreover, Adiposederived mesenchymal stem cells can easily be expanded in vitro, have anextensive self-renewal capacity, and are easily isolated in a laboratorysetting by differential sedimentation. Adipose derived mesenchymal stemcells can differentiate into various types of cells, includingadipocytes, osteoblasts, and chondrocytes (Morcos et al, 2015).

Adipose tissue is comprised of adipocytes and a heterogeneous set ofcell populations including endothelial cells, endothelial progenitorcells, pericytes, and erythrocytes that surround and support them, whichupon isolation are termed the stromal vascular fraction. In order toisolate adipose derived mesenchymal stem cells, adipose cells areharvested and then minced and digested by collagenase type II. Then thestromal vascular fraction is separated by centrifugation as it has ahigher density than the adipocytes. Later on, isolate adipose derivedmesenchymal stem cells are isolated from the stromal vascular fractionby plastic adherence in culture, which can easily be cultured andexpanded in vitro. Moreover, isolated adipose derived mesenchymal stemcells can be cryopreserved in a media of serum and dimethyl sulfoxidewithout losing their ability to differentiate and proliferate (Morcos etal, 2015).

Several approaches for isolating adipose derived mesenchymal stem cellsfrom an adipose tissue, and for using the isolated adipose derivedmesenchymal stem cells for soft tissue or bone tissue regeneration, areknown in the art.

One approach involves the use of enzymes. For example, United Statespatent application publication No. US 2008/0095750 A1 describes aprocedure for isolating adult stem cells from human liposuction tissue,the procedure comprises collagenase digestion of the liposuction tissue,differential centrifugation and expansion in culture. Similarly, UnitedStates patent application publication No. US 2015/0037289 A1 describes amethod for preparing a stem cells preparation for treating a bone,ligament, tendon or cartilage injury in an animal, the methodcomprising: collecting adipose tissue from a source animal, contactingthe adipose tissue with an enzyme preparation, including collagenase andprotease, that digests fat and connective tissues contained in theadipose tissue, while preserving the stem cells in the adipose tissue,and collecting the stem cells. Afterwards, blood is collected from thesame source animal and used for the preparation of platelet rich plasma,which is mixed with the collected stem cells in order to activate thestem cells.

Enzymes such as collagenase are typically used to dissolve the bonds inthe collagen that hold together the adipose tissue (see, for example,Zuk, et al. Mol Biol Cell. 2002; 13: 4279-4295; Zuk, et al. Tissue Eng.2001; 7: 211-228). While collagenase is effective, it can be unsuitablefor preparing stem cells for the following reasons:

-   -   enzyme treatment results in a high level of cell death, thereby        reducing numbers of isolated stem cells and resulting in more        cellular debris;    -   enzymes may damage and destroy unique cell types;    -   contamination of isolated stem cells with enzymes may make them        unsuitable for transplantation; and    -   regulatory bodies may consider that the use of enzymes in the        isolation of stem cells results in a cellular product requiring        drug approval.

Another approach is in vitro culturing of isolated adipose derivedmesenchymal stem cells with specific growth media for differentiatingthe stem cells for a desired cell type. For example, U.S. Pat. No.6,777,231 B1 describes inter alia obtaining raw liposuction aspiratefrom a patient, rinsing the liposuction aspirate, digesting withcollagenase, centrifuging, treating a cellular pellet with anerythrocyte-lysing solution, and isolating the obtained stem cells bycentrifugation. Then, a population of isolated adipose derived stemcells was cultured at high density in a chondrogenic medium for severalweeks. In addition, a population of isolated adipose derived stem cellswas cultured until near confluence and then exposed to an osteogeneicmedium for several weeks.

Yet another approach involves the use of wave energy. For example,United States patent application publication No. US 2006/0051865 A1describes methods for isolating cells from adipose tissue that havepotential to differentiate into cells of mesenchymal origin, the methodscomprise subjecting adipose tissue to an electromagnetic, sonic, orother wave energy source, and centrifuging the tissue to form a pelletcomprising stem cells. Also United States patent application publicationNo. US 2015/0231244 A1 describes an adipose-derived stem cell processingsystem comprising inter alia an ultrasonic generator configured toexcite a raw adipose tissue with ultrasonic energy for isolatingadipose-derived stem cells from the raw adipose tissue.

The currently known methods, devices and systems for isolating adiposederived mesenchymal stem cells from an adipose tissue for soft tissue orbone tissue regeneration—are cumbersome, expensive and complicated,sometimes involving the use of enzymes and occasionally take longperiods of time.

SUMMARY OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

According to one aspect of the present invention, there is provided adevice configured to mechanically remove a fat phase from at least oneadipose tissue, the device comprising a first pressing element and asecond pressing element.

According to one embodiment, the device further comprises a fat phaseseparating element.

According to another embodiment, the fat phase separating element is asucking element.

According to yet another embodiment, the fat phase separating element isa drainage element.

According to still another embodiment, the device further comprises atleast one perforating element.

According to a further embodiment, at least one of the pressing elementsis substantially flat.

According to yet a further embodiment, at least one of the pressingelements is substantially cylindrical.

According to still a further embodiment, the first pressing element hasa bowl-like structure comprising a base and at least one wall, and thesecond pressing element has a bowl-like structure comprising a base andat least one wall, wherein the bowl-like second pressing element isconfigured to be accommodated in a space defined by the base and the atleast one wall of the bowl-like first pressing element.

According to an additional embodiment, the device comprising a bowl-likefirst pressing element and a bowl-like second pressing element, furthercomprises a pressing and perforating unit comprising a frame enclosing alattice of a plurality of crossed strips defining a plurality of latticespaces; the frame and lattice comprising a first surface configured tobe in contact with at least one adipose tissue and a second surface; thepressing and perforating unit further comprising at least oneperforating member comprising a tip pointing towards the first surfaceand configured to perforate at least one adipose tissue by extendingbeyond the first surface, a leg attached to the tip and a resilientconnector connecting the tip to a strip; wherein the pressing andperforating unit is configured to be placed on the base of the bowl-likefirst pressing element.

According to another aspect of the present invention, there is provideda method for preparing a preparation comprising a fat-depleted adiposetissue, the method comprising: mechanically removing a fat phase from atleast one adipose tissue.

According to one embodiment, the mechanically removing a fat phase fromat least one adipose tissue comprises: homogenizing the at least oneadipose tissue to provide a homogenate, and extracting a fat-depletedadipose tissue from the homogenate.

According to another embodiment, the mechanically removing a fat phasefrom at least one adipose tissue comprises: pressing the at least oneadipose tissue.

According to yet another embodiment, the pressing at least one adiposetissue comprises: pressing the at least one adipose tissue between afirst pressing element and a second pressing element.

According to still another embodiment, the method further comprises:separating the fat phase from the fat-depleted adipose tissue.

According to a further embodiment, the method further comprises:perforating the at least one adipose tissue.

According to yet a further embodiment, the method further comprises:adding a preparation which is configured to enhance proliferation anddifferentiation of mesenchymal stem cells.

According to still a further embodiment, the method further comprises:mixing with at least one type of bone tissue.

According to an additional embodiment, the method further comprises:mixing with at least one type of bone substitute.

According to a further aspect of the present invention, there isprovided a preparation comprising a fat-depleted adipose tissue, whereinthe fat-depleted adipose tissue is prepared by mechanically removing afat phase from at least one adipose tissue.

According to one embodiment, the preparation further comprises apreparation which is configured to enhance proliferation anddifferentiation of mesenchymal stem cells.

According to another embodiment, the preparation further comprises atleast one type of bone tissue.

According to yet another embodiment, the preparation further comprisesat least one type of bone substitute.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments are herein described, by way of example only, with referenceto the accompanying drawings. With specific reference now to thedrawings in detail, it is stressed that the particulars shown are by wayof example and for purposes of illustrative discussion of the preferredembodiments, and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of the embodiments. In this regard, noattempt is made to show structural details in more detail than isnecessary for a fundamental understanding, the description taken withthe drawings making apparent to those skilled in the art how severalforms may be embodied in practice.

In the drawings:

FIG. 1 schematically illustrates, according to an exemplary embodiment,a fat phase removing device, comprising a first pressing element and asecond pressing element.

FIG. 2 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device, further comprisinga drainage element.

FIG. 3 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device further comprising aperforating element attached to the first pressing element.

FIG. 4 schematically illustrates, according to an exemplary embodiment,a cross section view of a fat phase removing device further comprising aperforating element attached to the second pressing element.

FIG. 5 schematically illustrates, according to an exemplary embodiment,a cross section view of a fat phase removing device further comprising aperforating element comprising at least one tooth-like structure,attached to the first pressing element, and a perforating elementcomprising at least one tooth-like structure, attached to the secondpressing element.

FIG. 6 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising asubstantially flat first pressing element and a substantiallycylindrical second pressing element.

FIG. 7 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising asubstantially flat first pressing element and a substantiallycylindrical second pressing element, wherein the substantially flatfirst pressing element further comprises a drainage element.

FIG. 8 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising asubstantially flat first pressing element and a substantiallycylindrical second pressing element, wherein the substantially flatfirst pressing element further comprises a drainage element and aperforating element, comprising at least one tooth-like structure,attached to the substantially flat first pressing element.

FIG. 9 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising asubstantially flat first pressing element and a substantiallycylindrical second pressing element, wherein the substantially flatfirst pressing element further comprises a drainage element, and thesubstantially cylindrical second pressing element further comprises aperforating element.

FIG. 10 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising asubstantially flat first pressing element and a substantiallycylindrical second pressing element, wherein the substantially flatfirst pressing element further comprises a drainage element, and aperforating element comprising at least one tooth-like structure, andthe substantially cylindrical second pressing element further comprisesa perforating element comprising at least one tooth-like structure.

FIG. 11 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising asubstantially cylindrical first pressing element and a substantiallycylindrical second pressing element.

FIG. 12 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising asubstantially cylindrical first pressing element and a substantiallycylindrical second pressing element, wherein the substantiallycylindrical second pressing element further comprises a perforatingelement comprising at least one tooth-like structure.

FIG. 13 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising asubstantially cylindrical first pressing element and a substantiallycylindrical second pressing element, wherein the substantiallycylindrical first pressing element further comprises a perforatingelement comprising at least one tooth-like structure, and thesubstantially cylindrical second pressing element further comprises aperforating element comprising at least one tooth-like structure.

FIG. 14 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising abowl-like first pressing element and a bowl-like second pressingelement.

FIG. 15 schematically illustrates, according to an exemplary embodiment,a perspective view of a pressing and perforating unit, configured topress and perforate at least one adipose tissue.

FIG. 16 schematically illustrates, according to an exemplary embodiment,a cross-section view of a pressing and perforating unit, in a relaxedstate.

FIG. 17 schematically illustrates according to an exemplary embodiment,a cross-section view of a pressing and perforating unit, in a pressedstate.

FIG. 18 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device comprising abowl-like first pressing element and a bowl-like second pressingelement, in combination with a pressing and perforating unit.

FIG. 19 schematically illustrates, according to an exemplary embodiment,a perspective view of a pressure exerting device.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before explaining at least one embodiment in detail, it is to beunderstood that the invention is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting. In discussion of thevarious figures described herein below, like numbers refer to likeparts. The drawings are generally not to scale.

For clarity, non-essential elements were omitted from some of thedrawings.

It is an object of the present invention to provide a simple andinexpensive device and method for providing within a short period oftime a preparation derived from an adipose tissue which is suitable fortransplantation or injection in a patient with a need for soft tissue orbone tissue regeneration.

One aim of the present invention is to provide a device for preparing apreparation comprising a fat-depleted adipose tissue.

Another aim of the present invention is to provide a method forpreparing a preparation comprising a fat-depleted adipose tissue.

Yet another aim of the present invention is to provide a preparationcomprising a fat-depleted adipose tissue, configured to be transplantedor injected in a patient with a need for soft tissue or boneregeneration.

The term “adipose tissue” as disclosed herein refers to a fat tissueobtained from an animal, the adipose tissue comprising inter aliaadipocytes and mesenchymal stem cells.

The term “animal” as disclosed herein refers to any eukaryoticmulticellular organism having an adipose tissue, including human being.

The term “patient” as disclosed herein refers to an animal from which anadipose tissue may be provided, and/or with a need for soft tissue orbone regeneration.

The term “fat phase” as disclosed herein refers to a portion of anadipose tissue comprising, according to some embodiments, intact adiposecells, namely adipocytes. According to some other embodiments, the fatphase may further comprise triglycerides. According to some additionalembodiments, the fat phase may further comprise cholesteryl ester.According to still additional embodiment, the fat phase may furthercomprise adipocyte debris.

A “fat-depleted adipose tissue” as disclosed herein refers to an adiposetissue from which at least part of a fat phase was removed by mechanicalmeans. According to some embodiments, a preparation comprising thefat-depleted adipose tissue is configured to be transplanted or injectedin a patient with a need for soft tissue regeneration. According to someother embodiments, a preparation comprising the fat-depleted adiposetissue is configured to be transplanted or injected in a patient with aneed for bone regeneration. It should be noted though, that any use of apreparation comprising a fat-depleted adipose tissue prepared accordingto embodiments of the present invention is under the scope of thepresent invention.

According to one aspect of the present invention, a device for preparinga preparation comprising a fat-depleted adipose tissue, occasionallytermed hereinafter as “fat phase removing device”, or “the device”, isprovided.

According to another aspect of the present invention, a method forpreparing a preparation comprising a fat-depleted adipose tissue,occasionally termed hereinafter as “the method”, is provided.

According to yet another aspect of the present invention, a preparationcomprising a fat-depleted adipose tissue, configured to be transplantedor injected in a patient with a need for soft tissue or boneregeneration, occasionally term hereinafter as “the preparation”, isprovided.

FIG. 1 schematically illustrates, according to an exemplary embodiment,a fat phase removing device 100, comprising a first pressing element 10and a second pressing element 20. The device 100 is configured tomechanically remove a fat phase from at least one adipose tissue 500.Furthermore, the device 100 is configured to press at least one adiposetissue 500 in between the first pressing element 10 and the secondpressing element 20, in a manner that forces out at least part of a fatphase from the at least one adipose tissue 500. Pressing at least oneadipose tissue 500 in between the first pressing element 10 and thesecond pressing element 20 may crush adipose cells, for example largeadipose cells, present in the at least one adipose tissue 500. As aresult, at least part of a fat phase is forced out from the at least oneadipose tissue 500.

According to one embodiment, the first pressing element 10 isstationary, and the second pressing element 20 moves towards the firstpressing element, while pressing at least one adipose tissue 500 inbetween. According to another embodiment, the second pressing element 20is stationary and the first pressing element 10 moves towards the secondpressing element 20, while pressing at least one adipose tissue 500 inbetween. According to a further embodiment, the first pressing element10 and the second pressing element 20 both move one towards the other,while pressing at least one adipose tissue 500 in between.

According to one embodiment, the first pressing element 10 is above thesecond pressing element 20 (not shown). According to another embodiment,the first pressing element 10 is below the second pressing element 20,as illustrated in FIG. 1. According to a further embodiment, the firstpressing element 10 is aside the second pressing element 20. Accordingto ant one of the aforementioned embodiments, at least one adiposetissue 500 is placed in between the first pressing element 10 and thesecond pressing element 20 during the pressing of the at least oneadipose tissue 500.

According to some embodiments, the first pressing element 10 and thesecond pressing element 20 are substantially flat, as illustrated forexample in FIG. 1. Thus, a first pressing element 10 is for example atable surface, and a second pressing element 20 is for exampleweight-like element. It should be noted though that the first pressingelement 10 and the second pressing element 20 may have any structurewhich enables pressing of at least one adipose tissue 500.

According to one embodiment, the first pressing element 10 and/or thesecond pressing element 20, which moves during the pressing of at leastone adipose piece 500, is manually moved. For example but not limitedto, the moving pressing element 10 or 20 may be held by a hand of a userand moved towards the other pressing element 20 or 10 during thepressing of at least one adipose tissue 500.

According to another embodiment, the first pressing element 10 and/orthe second pressing element 20, which moves during the pressing of atleast one adipose piece 500, is moved by a mechanical mechanism, forexample but not limited to, a motor, an electrical motor, a manuallyoperated mechanical mechanism, and the like.

According to yet another embodiment, any combination of the ways formoving a pressing element, in case where both the first pressing element10 and the second pressing element 20 are moving, is under the scope ofthe present invention. For example, the first pressing element 10 andthe second pressing element 20 may be manually moved; the first pressingelement 10 and the second pressing element 20 may be moved by amechanical mechanism; the first pressing element 10 may be manuallymoved and the second pressing element 20 may be moved by a mechanicalmechanism, or vice versa.

According to an additional embodiment, the first pressing element 10 andthe second pressing element 20 are configured to apply a controlledpressure on the at least one adipose tissue 500 during pressing. Thepressure level affects the amount of fat phase removed from the at leastone adipose tissue 500 in one hand, and affects the quality of theobtained fat-depleted adipose tissue on the other hand. Accordingly, acertain minimal pressure level should be applied on the at least oneadipose tissue 500 in order to enable removal of fat phase from the atleast one adipose tissue. On the other hand, a certain maximal pressurelevel should be applied on the at least one adipose tissue 500 in orderto avoid destruction of the at least one adipose tissue, rendering theobtained fat-depleted adipose tissue useless. Thus, according to thisembodiment, the fat phase removing device 100 further comprises apressure control element configured to control the pressure which isapplied on the at least one adipose tissue 500 by the first pressingelement 10 and the second pressing element 20.

According to one embodiment, the first pressing element 10 and thesecond pressing element 20 are configured to become in contact whenpressing at least one adipose tissue 500 in between. According to apreferred embodiment, during pressing of the at least one adipose tissue500, a gap in a certain size is maintained between the first pressingelement 10 and the second pressing element 20, for example in order toavoid over-pressing or crushing of the at least one adipose tissue 500.The size of the gap maintained between the first pressing element 10 andthe second pressing element 20, during pressing of at least one adiposetissue 500, is correlated to the size of the at least one adipose tissue500 being pressed. According to one embodiment, the gap is in the rangeof substantially 0-500 μm. According to another embodiment, the gap isin the range of substantially 0-300 According to yet another embodiment,the gap is in the size of substantially 150 It should be noted, though,that a gap maintained in any size between the first pressing element 10and the second pressing element 20 is under the scope of the presentinvention. Thus, according to this embodiment, the fat phase removingdevice 100 further comprises a distance controlling element, configureto control the distance of movement of the first pressing element 10and/or the second pressing element 20 towards each other, in order tomaintain a gap in a predetermined size between the first pressingelement 10 and the second pressing element 20.

According to another embodiment, the first pressing element 10 and/orthe second pressing element 20, which moves during the pressing of atleast one adipose tissue 500, comprises at least one weight-like elementhaving a known weight value. Thus, according to an exemplary embodiment,the first pressing element 10 is stationary. At least one adipose tissue500 is placed on the first pressing element 10, and the second pressingelement 20 comprises at least one weight-like element, which presses theat least one adipose tissue 500 from above. According to thisembodiment, controlling the pressure applied on the at least one adiposetissue 500 is achieved by using at least one weight-like element with adesired weight value.

According to yet another embodiment, the first pressing element 10and/or the second pressing element 20, which moves during the pressingof at least one adipose tissue 500, is configured to move in a certainvelocity. The velocity of the movement of a moving pressing elementaffects the amount of fat phase removed from the at least one adiposetissue 500 in one hand, and affects the quality of the obtainedfat-depleted adipose tissue on the other hand. Thus, according to thisembodiment, the fat phase removing device 100 further comprises avelocity control element configured to control the velocity in which amoving pressing element moves when pressing at least one adipose tissue500.

According to still another embodiment, the fat phase removing device 100is configured to press the at least one adipose tissue 500 for a certainperiod of time. The period of time during which the at least one adiposetissue 500 is pressed affects the amount of fat phase removed from theat least one adipose tissue 500 in one hand, and affects the quality ofthe obtained fat-depleted adipose tissue on the other hand. Thus,according to this embodiment, the fat phase removing device 100 furthercomprises a timer configured to control the period of time during whichthe at least one adipose tissue 500 is pressed.

According to an additional embodiment, the fat phase removing device 100is configured to press the at least one adipose tissue 500 under acontrolled temperature. The temperature under which the at least oneadipose tissue 500 is pressed affects the amount of fat phase removedfrom the at least one adipose tissue 500 in one hand, and affects thequality of the obtained fat-depleted adipose tissue on the other hand.For example, increasing the temperature renders fatty material in the atleast one adipose tissue 500 more fluid and less solid, thus increasingthe amount of fat phase removed from the at least one adipose tissue 500during pressing. On the other hand, increasing the temperature too muchmay threaten the viability of cells present in the fat-depleted adiposetissue. According to some exemplary embodiments, the fat phase removingdevice 100 is configured to press the at least one adipose tissue 500under, for example but not limited to, ambient temperature, atemperature in the range of substantially 18-28° C., and preferably atemperature in the range of substantially 37-42° C. Thus, according tothis embodiment, the fat phase removing device 100 further comprises atemperature control element configured to control the temperature underwhich the at least one adipose tissue 500 is pressed.

According to some embodiments, the fat phase removing device 100 furthercomprises a fat phase separating element. According to one embodiment,the fat phase separating element is separated from the first pressingelement 10 and/or the second pressing element 20. According to anotherembodiment, the fat phase separating element is attached to the firstpressing element 10 and/or the second pressing element 20. According toyet another embodiment, the fat phase separating element is part of thefirst pressing element 10 and/or the second pressing element 20.

According to some embodiments, the fat phase separating element isconfigured to separate the fat phase from the fat-depleted adiposetissue during the pressing of the at least one adipose tissue 500 withthe fat phase removing device 100. According to some other embodiments,the fat phase separating element is configured to separate the fat phasefrom the fat-depleted adipose tissue after the pressing of the at leastone adipose tissue 500 with the fat phase removing device 100.

According to one embodiment, the fat phase separating element is asucking element configured to suck a fat phase, thus separating the fatphase from the fat-depleted adipose tissue. Examples of a suckingelement include, but not limited to, a syringe-like element, anaspirator-like element, and a catheter fluidically connected to a vacuumpump.

According to another embodiment, the fat phase separating element is adrainage element, separating the fat phase from the fat-free adiposetissue by draining the fat phase away. Examples of a drainage elementinclude, but not limited to, a drainage slot on a surface of a pressingelement 10 and/or 20 configured to allow flow of a fat phase away fromthe pressed at least one adipose tissue 500 during or after pressing.According to one embodiment, the first pressing element 10 comprises adrainage element. According to another embodiment, the second pressingelement 20 comprises a drainage element. According to yet anotherembodiment, both the first pressing element 10 and the second pressingelement 20 comprise a drainage element.

FIG. 2 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100, furthercomprising a drainage element 30. According to the exemplary embodimentillustrated in FIG. 2, the drainage element 30 is a drainage slot in theperiphery of the first pressing element 10.

According to some embodiments, the fat phase removing device 100 furthercomprises at least one perforating element. The at least one perforatingelement is configured to perforate or incise the at least one adiposetissue 500 in a manner that facilitates exit of a fat phase from the atleast one adipose tissue 500 during pressing of the at least one adiposetissue 500.

According to some embodiments, the perforating element is separated fromthe first pressing element 10 and/or the second pressing element 20. Anyperforating element known in the art, which is configured to perforateor incise at least one adipose tissue 500 is under the scope of thepresent invention, for example but not limited to, a needle, a scalpel,tweezers, an element comprising a plurality of tooth-like structuresresembling a comb or a brush, and the like.

According to some other embodiments, the at least one perforatingelement is attached to the first pressing element 10 and/or the secondpressing element 20 in a manner that allows perforation or incision ofthe at least one adipose tissue 500 during pressing with the fat phaseremoving device 100. It should be noted in this context that the term“attached” is defined as physically attached, for example by gluing orwelding, or as being part of the fat phase removing device 100, forexample created during casting of a pressing element 10 and/or 20 madeof metal or plastic. An example for illustrative purposes only of aperforating element attached to the first pressing element 10 and/or thesecond pressing element 20 is an at least one tooth-like structureattached to the surface of the first pressing element 10 and/or thesecond pressing element 20.

FIG. 3 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 furthercomprising a perforating element 140 attached to the first pressingelement 10. The perforating element 140 may be of any type as describedabove. According to the exemplary embodiment illustrated in FIG. 3, theperforating element 140 comprises at least one tooth-like structure 145.During the pressing of the at least one adipose tissue 500 between thefirst pressing element 10 and the second pressing element 20, theperforating element 140 which is attached to the surface of the firstpressing element 10 is configured to perforate the at least one adiposetissue 500 and facilitate exit of a fat phase from the at least oneadipose tissue 500 during pressing.

FIG. 4 schematically illustrates, according to an exemplary embodiment,a cross section view of a fat phase removing device 100 furthercomprising a perforating element 240 attached to the second pressingelement 20. According to the exemplary embodiment illustrated in FIG. 4,the perforating element 240 comprises at least one tooth-like structure245. During the pressing of the at least one adipose tissue 500 betweenthe first pressing element 10 and the second pressing element 20, theperforating element 240 which is attached to the surface of the secondpressing element 20 is configured to perforate the at least one adiposetissue 500 and facilitate exit of a fat phase from the at least oneadipose tissue 500 during pressing.

FIG. 5 schematically illustrates, according to an exemplary embodiment,a cross section view of a fat phase removing device 100 furthercomprising a perforating element 140 comprising at least one tooth-likestructure 145, attached to the first pressing element 10, and aperforating element 240 comprising at least one tooth-like structure245, attached to the second pressing element 20. During the pressing ofthe at least one adipose tissue 500 between the first pressing element10 and the second pressing element 20, both perforating elements 140 and240 are configured to perforate the at least one adipose tissue 500 andfacilitate exit of a fat phase from the at least one adipose tissue 500during pressing.

According to the embodiments illustrated in FIGS. 1-5, the firstpressing element 10 and the second pressing element 20 are substantiallyflat. According to some other embodiments, at least one of the pressingelements is substantially cylindrical, namely either the first pressingelement 10 and/or the second pressing element are substantiallycylindrical. According to some further embodiments, at least one of thepressing elements is substantially flat. According to some additionalembodiments, at least one of the pressing elements is substantiallycylindrical. Nevertheless, it should be emphasized again that anystructure of the first pressing element 10 and the second pressingelement 20, which is configured to enable pressing of at least oneadipose tissue 500, is under the scope of the present invention.

FIG. 6 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising asubstantially flat first pressing element 10 and a substantiallycylindrical second pressing element 60, configured to press at least oneadipose tissue 500 in between the substantially flat first pressingelement 10 and the substantially cylindrical second pressing element 60,in a manner that forces out at least part of a fat phase from the atleast one adipose tissue 500. The substantially cylindrical secondpressing element 60 is configured to roll over the at least one adiposetissue 500 while pressing the at least one adipose tissue 500.

FIG. 7 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising asubstantially flat first pressing element 10 and a substantiallycylindrical second pressing element 60, wherein the substantially flatfirst pressing element 10 further comprises a drainage element 30.According to the exemplary embodiment illustrated in FIG. 7, thedrainage element 30 is a drainage slot in the periphery of thesubstantially flat first pressing element 10.

FIG. 8 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising asubstantially flat first pressing element 10 and a substantiallycylindrical second pressing element 60, wherein the substantially flatfirst pressing element 10 further comprises a drainage element 30 and aperforating element 140, comprising at least one tooth-like structure145, attached to the substantially flat first pressing element 10.

FIG. 9 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising asubstantially flat first pressing element 10 and a substantiallycylindrical second pressing element 60, wherein the substantially flatfirst pressing element 10 further comprises a drainage element 30, andthe substantially cylindrical second pressing element 60 furthercomprises a perforating element 640. As described above, the perforatingelement 640 may be of any type. According to the exemplary embodimentillustrated in FIG. 9, the perforating element 640 comprises at leastone tooth-like structure 645.

FIG. 10 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising asubstantially flat first pressing element 10 and a substantiallycylindrical second pressing element 60, wherein the substantially flatfirst pressing element 10 further comprises a drainage element 30, and aperforating element 140 comprising at least one tooth-like structure145, and the substantially cylindrical second pressing element 60further comprises a perforating element 640 comprising at least onetooth-like structure 645.

According to the embodiments illustrated in FIG. 6-10, one of the twopressing elements is substantially flat and the other pressing elementis substantially cylindrical. Several embodiments regarding the movementof the pressing elements—one relative to the other, are as follows:According to one embodiment, the substantially flat pressing element isconfigured to be stationary and the substantially cylindrical pressingelement is configured to roll along the surface of the substantiallyflat pressing element during the pressing of the at least one adiposetissue 500. According to another embodiment, the substantiallycylindrical pressing element is configured to be stationary and rollalong the surface of the substantially flat pressing element, whichmoves in a manner that allows rolling of the substantially cylindricalpressing element along the surface of the substantially flat pressingelement, during the pressing of the at least one adipose tissue 500.According to yet another embodiment, both the substantially cylindricalpressing element and the substantially flat pressing element areconfigured to move in a manner that allows rolling of the substantiallycylindrical pressing element along the surface of the substantially flatpressing element, during the pressing of the at least one adipose tissue500.

According to some additional embodiments, both the first pressingelement and the second pressing element are substantially cylindrical.According to these embodiments, both substantially cylindrical pressingelements are configured to roll one relative to the other in a mannerthat presses the at least one adipose tissue 500.

FIG. 11 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising asubstantially cylindrical first pressing element 70 and a substantiallycylindrical second pressing element 60, both configured to press atleast one adipose tissue 500 in between.

FIG. 12 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising asubstantially cylindrical first pressing element 70 and a substantiallycylindrical second pressing element 60, wherein the substantiallycylindrical second pressing element 60 further comprises a perforatingelement 640 comprising at least one tooth-like structure 645.

FIG. 13 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising asubstantially cylindrical first pressing element 70 and a substantiallycylindrical second pressing element 60, wherein the substantiallycylindrical first pressing element 70 further comprises a perforatingelement 740 comprising at least one tooth-like structure 745, and thesubstantially cylindrical second pressing element 60 further comprises aperforating element 640 comprising at least one tooth-like structure645.

FIG. 14 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising abowl-like first pressing element 80 and a bowl-like second pressingelement 90.

The bowl-like first pressing element 80 comprises a base 802 configuredto allow the placement of at least one adipose tissue 500 on the base802. The base may have any structure and configuration. According to thepreferred exemplary embodiment illustrated in FIG. 14, the base 802 ofthe bowl-like first pressing element 80 is flat. The bowl-like firstpressing element further comprises at least one wall 804, when thenumber of walls 804 depends on the structure of the base 802. Inembodiments according to which the base 802 is circular, there is onewall 804 extending from the perimeter of the circular base 804; whereasin embodiments according to which the base 802 is quadrangular, thereare four walls 804, when each wall 804 extends from an edge of thequadrangular base 802. The base 802 and the at least one wall 804 definea space 806. According to some embodiments, the bowl-like first pressingelement 80 further comprises a drainage element 30.

The bowl-like second pressing element 90 comprises a base 902 and atleast one wall 904, which both define a space 906. The bowl-like secondpressing element is configured to be accommodated in the space 806 ofthe bowl-like first pressing element 80. Thus, any structure andconfiguration of the bowl-like second pressing element 90, inter alia interms of the structure of the base 902 and at least one wall 904, whichallow accommodation of the bowl-like second pressing element 90 in thespace 806 of the bowl-like first pressing element 80, is under the scopeof the present invention.

FIG. 15 schematically illustrates, according to an exemplary embodiment,a perspective view of a pressing and perforating unit 1000, configuredto press and perforate at least one adipose tissue 500. According tosome embodiment, the pressing and perforating unit is configured toperforate and press at least one adipose tissue independently. Accordingto a preferred embodiment, the pressing and perforating unit 1000 isconfigured to perforate and press at least one adipose tissue 500 incombination with a fat phase removing device 100. According to oneembodiment, illustrated in FIG. 15, the pressing and perforating unit1000 comprises a frame 1010 enclosing a lattice of a plurality ofcrossed strips 1020 defining a plurality of lattice spaces 1030, and atleast one perforating member 1040 configured to extend from a latticespace 1030. According to some embodiments, the pressing and perforatingunit 1000 may be in a relaxed state. According to some otherembodiments, the pressing and perforating unit 1000 may be in a pressedstate.

FIG. 16 schematically illustrates, according to an exemplary embodiment,a cross-section view of a pressing and perforating unit 1000, in arelaxed state. The frame 1010 and lattice of a plurality of crossedstrips 1020 comprise a first surface 1012 and a second surface 1014. Thefirst surface 1012 is configured to be in contact with at least oneadipose tissue 500. The at least one perforating member 1040 comprises atip 1046 pointing towards the first surface 1012 and configured toperforate at least one adipose tissue 500. The at least one perforatingmember further comprises a leg 1044 attached to the tip 1046, and aresilient connector 1042 connecting the tip 1046 to a strip 1020.

According to some embodiments, the tip 1046 of the at least oneperforating member 1040 has an size and shape which are allowperforation of the at least one adipose tissue 500. According to oneembodiment, the tip 1046 is sharp as illustrated for example in FIG. 16.According to another embodiment, the width of the tip 1046 issubstantially 1 mm. According to yet another embodiment, the length ofthe tip is in the range of substantially 0.5-2 mm.

At a relaxed state, as illustrated in FIG. 16, according to a preferredembodiment, a tip 1046 of the at least one perforating member 1040 ispositioned at the level of the first surface 1012. According to anotherembodiment, the tip 1046 extends beyond the first surface 1012 (notshown), and according to yet another embodiment, the tip 1046 ispositioned below the first surface 1012. Furthermore, at the relaxedstate, according to a preferred embodiment, the leg 1044 extendsoutwards of the second surface 1014.

FIG. 17 schematically illustrates according to an exemplary embodiment,a cross-section view of a pressing and perforating unit 1000, in apressed state. According to some embodiments, the pressing andperforating unit 1000 is configured to be pressed towards the secondsurface 1014, as indicated with arrows 950, when the legs 1044 of the atleast one perforating member 1040 are placed on a surface (not shown).As a result, due to the contradictory force exerted by the surface onthe legs 1044, and the resiliency of the resilient connector 1042, thetips 1046 of the at least one perforating member 1040 are pushed towardsthe first surface 1012, as indicated with arrows 960, causing the tips1046 to extend beyond the first surface 1012. If at least one adiposetissue 500 is in contact with the first surface 1012, the tips 1046perforate the at least one adipose tissue 500 and allow exit of a fatphase from the at least one adipose tissue 500 during the pressing. Whenthe pressure in direction 950 is relieved, due to the resiliency of theresilient connector 1042 the at least one perforating member 1040returns to the relaxed state, as illustrated in FIG. 16.

FIG. 18 schematically illustrates, according to an exemplary embodiment,a cross-section view of a fat phase removing device 100 comprising abowl-like first pressing element 80 and a bowl-like second pressingelement 90, in combination with a pressing and perforating unit 1000.The pressing and perforating unit 1000 stands on the base 802 of thebowl-like first pressing element 80, and at least one adipose tissue 500is in contact with the first surface 1012 of the pressing andperforating unit 1000. When the at least one adipose tissue 500 ispressed between the first surface 1012 of the pressing and perforatingunit 1000 and the base 902 of the bowl-like second pressing element 90,the at least one perforating member 1040 are pushed towards the firstsurface 1012, and the tips 1046 perforate the at least one adiposetissue 500, as described above. As a result of the pressing andperforation of the at least one adipose tissue 500, fat phase 510 exitsthe adipose tissue 500 and is collected on the base 802 of the bowl-likefirst pressing element 80, under the pressing and perforating unit 1000.Thus, the difference in height between the first surface 1012 of thepressing and perforating unit 1000 and the base 802 of the bowl-likefirst pressing element 80 serves as a drainage element in which the fatphase 510 is collected.

According to some embodiments, a pressure exerting device is provided.According to one general embodiment, the pressure exerting devicecomprises a body, a holder configured to hold a fat phase removingdevice 100, and a pushing press configured to provide pressure to thefat phase removing device 100 for pressing at least one adipose tissue.Furthermore, according to some additional embodiments, the pressureexerting device further comprises control elements as described above,for example but not limited to—a pressure control element, a velocitycontrol element, a timer, and a temperature control element.

It should be noted that the embodiments of the pressure exerting devicedisclosed herein should be understood as exemplary embodiments given fordemonstration purposes only, and that any pressure exerting device whichis configured to perform the functions disclosed herein is under thescope of the present invention.

FIG. 19 schematically illustrates, according to an exemplary embodiment,a perspective view of a pressure exerting device 2000. According to oneembodiment, the pressure exerting device 2000, illustrated in FIG. 19,is configured to provide pressure to a fat phase removing device 100comprising a bowl-like first pressing element 80 and a bowl-like secondpressing element 90, as illustrated for example in FIG. 14. According toanother embodiment, the pressure exerting device 2000, illustrated inFIG. 19, is configured to provide a pressure to a fat phase removingdevice 100 comprising a bowl-like first pressing element 80 and abowl-like second pressing element 90, in combination with a pressing andperforating unit 1000, as illustrated for example in FIG. 18.

According to some embodiments, the pressure exerting device 2000,illustrated in FIG. 19, comprises a body 2100 having a first part 2110and a second part 2120, a fat phase removing device holder 2200 attachedto the first part 2110 of the body 2100, and a pushing press 2300attached to second part 2120 of the body 2100. According to oneembodiment, the fat phase removing device holder 2200 is configured tohold a fat phase removing device 100 comprising a bowl-like firstpressing element 80 and a bowl-like second pressing element 90.According to another embodiment, the fat phase removing device holder2200 is configured to hold a fat phase removing device 100 comprising abowl-like first pressing element 80 and a bowl-like second pressingelement 90, in combination with a pressing and perforating unit 1000, asillustrated for example in FIG. 18. It should be noted that the pressureexerting device 2000, illustrated in FIG. 19, is only exemplary. Anypressure exerting device 2000, which is configured to exert pressure ona fat phase removing device 100 according to embodiments describedherein, is under the scope of the present invention.

According to yet another embodiment, the pushing press 2300, illustratedin FIG. 19, is configured to be accommodated in the space 906 of thebowl-like second pressing element 90. Furthermore, the pushing press2300 is configured to press the bowl-like second pressing element 90towards the bowl-like first pressing element 80, for pressing at leastone adipose tissue 500, as described above.

According to still another embodiment, the pressure exerting device 2000further comprises machinery for actuating the pushing press 2300 (notshown). According to yet a further embodiment, the pressure exertingdevice 2000 further comprises control elements as described above, forexample but not limited to—a pressure control element, a velocitycontrol element, a timer, and a temperature control element.

It should be noted that the configuration and orientation of thepressure exerting device 2000 as illustrated in FIG. 19 are onlyexemplary, and that other configurations and orientations of thepressure exerting device 2000 are under the scope of the presentinvention. For example, according to the embodiment illustrated in FIG.19, the fat phase removing device holder 2200 is positioned under thepushing press 2300. However, an opposite orientation in which the fatphase removing device holder 2200 is positioned above the pushing press2300 (not shown), is also under the scope of the present invention.

According to the embodiments illustrated in FIG. 1-19, the fat phaseremoving device 100 is configured to press at least one adipose tissue500 in order to allow exit of a fat phase from the at least one adiposetissue 500. The common feature of these embodiments is removal of a fatphase from at least one adipose tissue by exerting an external pressureon the at least one adipose tissue. It should be noted though that othertypes of pressure exerted on the at least one adipose tissue are underthe scope of the present invention, for example but not limited to,exerting a negative pressure on a fat phase of at least one adiposetissue.

According to another embodiment, of exerting a negative pressure on afat phase of at least one adipose tissue, there is provided a suckingelement configured to suck a fat phase from an adipose tissue. Thesucking action exerts negative pressure on the fat phase and as a resultthe fat phase is removed from the adipose tissue. Any sucking element,which is configured to suck a fat phase from an adipose tissue, is underthe scope of the present invention, for example but not limited to, asyringe, a cannula fluidically connected to a vacuum pump, and the like.

According to one embodiment, the sucking element is configured toindependently remove a fat phase from at least one adipose tissue. Forexample, according to some embodiments, a cannula fluidically connectedto a vacuum pump may penetrate the at least one adipose tissue, thusfacilitating access to a fat phase in the at least one adipose tissue,and removal of the fat phase, by the cannula fluidically connected to avacuum pump.

According to another embodiment, the sucking element is configured toremove a fat phase from at least one adipose tissue in combination withanother element, for example but not limited to, a perforating element.According to this embodiment, a perforating element as disclosed hereinis used to perforate at least one adipose tissue, thus facilitatingaccess for the sucking element to reach the fat phase of the at leastone adipose tissue and remove the fat phase from the at least oneadipose tissue.

According to another aspect of the present invention, there is provideda method for preparing a preparation comprising a fat-depleted adiposetissue, the method comprises mechanically removing a fat phase from atleast one adipose tissue.

At least one adipose tissue is provided from an animal. Any method knownin the art for providing an adipose tissue from an animal is under thescope of the present invention, for example but not limited to,aspiration of subcutaneous adipose tissue.

According to one embodiment, in the method for preparing a preparationcomprising a fat-depleted adipose tissue, the removing a fat phase fromat least one adipose tissue comprises homogenizing the adipose tissueand extracting a fat-depleted adipose tissue from the homogenate. Thehomogenization of the adipose tissue is performed, for example but notlimited to, by using a homogenizer. The extraction of the fat-freeadipose tissue from the homogenate is performed, for example but notlimited to, by centrifuging the homogenate. After centrifugation therephases are obtained—a lower aqueous phase comprising inter-cellularfluids, a middle cellular phase comprising cells derived from theadipose tissue, and an upper fatty phase comprising for exampletriglycerides. The middle cellular phase is a homogenized fat-depletedadipose tissue. Separation of the cellular phase from the lower phaseand the upper phase is performed by any method known in the art forseparating a cellular phase from a other phases, for example but notlimited to, aspiration of the cellular phase with a syringe, or pipette,or a cannula fluidically connected to a pump; and filtering through asubstrate configured to allow passage of an aqueous phase and a fattyphase, but not allow passage of a cellular phase. Examples of such asubstrate include, but not limited to, a membrane, a gauze and the like.

According to another embodiment, in the method for preparing apreparation comprising a fat-free adipose tissue, the removing a fatphase from at least one adipose tissue comprises pressing the at leastone adipose tissue.

According to yet another embodiment, in the method for preparing apreparation comprising a fat-free adipose tissue, the pressing the atleast one adipose tissue comprises pressing the at least one adiposetissue between a first pressing element and a second pressing element.Any embodiment known in the art of pressing the at least one adiposetissue between a first pressing element and a second pressing element,including embodiments disclosed herein, is under the scope of thepresent invention.

It should be noted that pressing the at least one adipose tissue byusing at least one hand palm of a user is also under the scope of thepresent invention, including but not limited to, pressing the at leastone adipose tissue with a hand palm against a surface, like a tablesurface; and pressing the at least one adipose tissue between to handpalms.

According to yet another embodiment, in the method for preparing apreparation comprising a fat-free adipose tissue, the pressing the atleast one adipose tissue comprises sucking the fat phase from the atleast one adipose tissue. Any embodiment known in the art of sucking afat phase from at least one adipose tissue, including embodimentsdisclosed herein, is under the scope of the present invention.

According to still another embodiment, the method for preparing apreparation comprising a fat-depleted adipose tissue further comprisesperforating the at least one adipose tissue. According to someembodiments, the perforating the at least one adipose tissue is beforethe removing a fat phase from at least one adipose tissue. According tosome other embodiments, the perforating the at least one adipose tissueis during the removing a fat phase from at least one adipose tissue. Anyembodiment known in the art of perforating at least one adipose tissue,before or during the removing of the fat phase from the adipose tissue,including embodiments disclosed herein, is under the scope of thepresent invention.

According to a further embodiment, the method for preparing apreparation comprising a fat-depleted adipose tissue further comprisescontrolling a pressure which is applied on the at least one adiposetissue during pressing. Any embodiment known in the art for controllinga pressure during pressing of at least one adipose tissue, includingembodiments disclosed herein, is under the scope of the presentinvention.

According to yet a further embodiment, the method for preparing apreparation comprising a fat-depleted adipose tissue further comprisescontrolling a velocity of a movement of any one of the first pressingelement and the second pressing element, or both, during the pressing ofthe at least one adipose tissue. Any embodiment known in the art forcontrolling the velocity of the movement of any one of the firstpressing element and the second pressing element, or both, includingembodiments disclosed herein, is under the scope of the presentinvention.

According to still a further embodiment, the method for preparing apreparation comprising a fat-depleted adipose tissue further comprisescontrolling a time period of pressing the at least one adipose tissue.Any embodiment known in the art of controlling the time period ofpressing at least one adipose tissue, including embodiments disclosedherein, is under the scope of the present invention.

According to an additional embodiment, the method for preparing apreparation comprising a fat-depleted adipose tissue further comprisescontrolling a temperature under which the at least one adipose tissue ispressed. Any embodiment known in the art of controlling the temperatureunder which the at least one adipose tissue is pressed, includingembodiments disclosed herein, is under the scope of the presentinvention.

According to one embodiment, the at least one adipose tissue is pressedunder ambient temperature. According to another embodiment, the at leastone adipose tissue is pressed under a temperature in the range ofsubstantially 18-28° C. According to a preferred embodiment, the atleast one adipose tissue is pressed under a temperature in the range ofsubstantially 37-40° C.

According to some embodiments, in the method for preparing a preparationcomprising a fat-depleted adipose tissue, the fat-depleted adiposetissue is configured, without any further manipulation or treatment, tobe transplanted or injected in a patient with a need for soft tissueregeneration, for example but not limited to, lack of soft tissue in themouse cavity and in areas of receding gums. Furthermore, thefat-depleted adipose tissue of the present invention is configured,without any further manipulation or treatment, to serve as a dentalregenerative membrane in dental surgery or oral surgery, or as a graftfor treating skin defects.

In order to increase the efficiency of soft tissue regeneration with thefat-free adipose tissue of the present invention, according to someembodiments, the method for preparing a preparation comprising afat-depleted adipose tissue further comprises adding a preparation richin growth factors which is configured to enhance proliferation anddifferentiation of mesenchymal stem cells. Any preparation known in theart, which is rich in growth factors that are configured to enhanceproliferation and differentiation of mesenchymal stem cells is under thescope of the present invention, for example but not limited to,platelet-rich fibrin, platelet-rich plasma, platelet-rich growth factor,plasma-rich growth factor, concentrated growth factors, and anycombination thereof.

According to one embodiment, the preparation rich in growth factors isadded to a fat-free adipose tissue which was pressed, for example butnot limited to, a fat-free adipose tissue which was pressed with thepressing device illustrated in FIGS. 1, 2, 6, 7, 11 and 14. According toa preferred embodiment, the preparation rich in growth factors is addedto a fat-free adipose tissue which was perforated and pressed, forexample but not limited to, a fat-free adipose tissue which wasperforated and pressed with the pressing device illustrated in FIGS.3-5, 8-10, 12-13 and 18. The perforation of the fat-free adipose tissuefacilitates entry of the preparation rich in growth factors into thefat-free adipose tissue, thus increasing the efficiency of the effect ofthe preparation rich in growth factors.

According to some embodiments, in the method for preparing a preparationcomprising a fat-depleted adipose tissue, the fat-depleted adiposetissue is configured to be transplanted or injected in a patient with aneed for bone tissue regeneration. Examples of situations in which thereis a need for bone tissue regeneration are detailed in the “Backgroundof the Invention”.

Thus, according to one embodiment, the method for preparing apreparation comprising a fat-depleted adipose tissue further comprisesadding at least one type of bone tissue to the fat-free adipose tissue.Any type of bone tissue known in the art is under the scope of thepresent invention. According to another embodiment, the method forpreparing a preparation comprising a fat-depleted adipose tissue furthercomprises adding at least one bone substitute to the fat-free adiposetissue. Any bone substitute known in the art is under the scope of thepresent invention. According to yet another embodiment, the method forpreparing a preparation comprising a fat-depleted adipose tissue furthercomprises adding at least one type of bone tissue and at least one bonesubstitute.

According to another embodiment, the method for preparing a preparationcomprising a fat-depleted adipose tissue further comprises adding apreparation rich in growth factors which is configured to enhanceproliferation and differentiation of mesenchymal stem cells, and addingat least one type of bone tissue to the fat-free adipose tissue. Theadding a preparation rich in growth factors which is configured toenhance proliferation and differentiation of mesenchymal stem cells isaccording to embodiments described above.

According to a preferred embodiment, the at least one type of bonetissue and/or the at least one bone substitute, with or without thepreparation rich in growth factors which is configured to enhanceproliferation and differentiation of mesenchymal stem cells, are mixedwith the fat-depleted adipose tissue, for example but not limited to, byusing a mixer. According to another preferred embodiment the mixing witha mixer is for a period of time in the range of substantially 5-10minutes. According to yet another preferred embodiment, the mixing isuntil the mixture is homogeneous. According to still another preferredembodiment, the mixing is until the mixture is semi-solid and suitablefor transplantation as a bone graft.

According to yet another aspect of the present invention, a preparationcomprising a fat-depleted adipose tissue, configured to be transplantedor injected in a patient with a need for soft tissue or boneregeneration, is provided.

According to one embodiment, the fat-depleted adipose tissue is preparedby mechanically removing a fat phase from at least one adipose tissue,according to any embodiment known in the art, including embodimentsdisclosed herein.

According to another embodiment, the preparation comprises afat-depleted adipose tissue and a preparation rich in growth factorswhich is configured to enhance proliferation and differentiation ofmesenchymal stem cells.

According to yet another embodiment, the preparation comprises afat-depleted adipose tissue and at least one type of bone tissue.

According to still another embodiment, the preparation comprises afat-depleted adipose tissue and at least one bone substitute.

According to a further embodiment, the preparation comprises afat-depleted adipose tissue, at least one type of bone tissue and atleast one bone substitute.

According to yet a further embodiment, the preparation comprises afat-depleted adipose tissue, a preparation rich in growth factors whichis configured to enhance proliferation and differentiation ofmesenchymal stem cells and at least one type of bone tissue.

According to still a further embodiment, the preparation comprises afat-depleted adipose tissue, a preparation rich in growth factors whichis configured to enhance proliferation and differentiation ofmesenchymal stem cells and at least one bone substitute.

According to an additional embodiment, the preparation comprises afat-depleted adipose tissue, a preparation rich in growth factors whichis configured to enhance proliferation and differentiation ofmesenchymal stem cells, at least one type of bone tissue, and at leastone bone substitute.

It should be noted that any one of the components of the preparationconfigured to be transplanted or injected in a patient with a need forsoft tissue or bone regeneration, according to embodiments disclosedherein, namely the fat-depleted adipose tissue, the preparation rich ingrowth factors which is configured to enhance proliferation anddifferentiation of mesenchymal stem cells, the at least one type of bonetissue, and the at least one bone substitute—are prepared according toembodiments known in art, including embodiments disclosed herein.

It should be further noted, that usage of the device for preparing apreparation comprising a fat-depleted adipose tissue of the presentinvention, and the method for preparing a preparation comprising afat-depleted adipose tissue of the present invention—significantlyshorten in time and simplify the preparation of a preparation configuredto be transplanted or injected in a patient with a need for soft tissueor bone regeneration. The currently available methods involve forexample enzymatic treatment of the adipose tissue, which burdens thepreparation process; or require prolonged incubation times, whichprolong the preparation time in the scale of days and weeks.Furthermore, the currently available methods require the usage ofspecial laboratory equipment, for example incubators. On the other hand,the present invention provides a device which is very simple to operateand less expensive. In addition, the present invention provides a muchfaster method for preparing the preparation. For example, the method forpreparing a preparation configured to be transplanted or injected in apatient with a need for soft tissue regeneration lasts for up tosubstantially half an hour, and the method for preparing a preparationconfigured to be transplanted or injected in a patient with a need forbone regeneration lasts for up to substantially an hour. Furthermore,the preparation provided by the present invention shortens theregeneration and repair time of a soft tissue or a bone followingtransplantation or injection, as well as increases the success rate ofclinical procedures involving the usage of the preparation of thepresent invention.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub combination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

1. A device configured to mechanically remove a fat phase from at leastone adipose tissue, the device comprising a first pressing element and asecond pressing element.
 2. The device according to claim 1, furthercomprising a fat phase separating element.
 3. The device according toclaim 2, wherein the fat phase separating element is a sucking element.4. The device according to claim 2, wherein the fat phase separatingelement is a drainage element.
 5. The device according to claim 4,further comprising at least one perforating element.
 6. The deviceaccording to claim 4, wherein at least one of the pressing elements issubstantially flat.
 7. The device according to claim 4, wherein at leastone of the pressing elements is substantially cylindrical.
 8. The deviceaccording to claim 1, wherein the first pressing element has a bowl-likestructure comprising a base and at least one wall, and the secondpressing element has a bowl-like structure comprising a base and atleast one wall, wherein the bowl-like second pressing element isconfigured to be accommodated in a space defined by the base and the atleast one wall of the bowl-like first pressing element.
 9. The deviceaccording to claim 8, further comprising a pressing and perforating unitcomprising a frame enclosing a lattice of a plurality of crossed stripsdefining a plurality of lattice spaces; the frame and lattice comprisinga first surface configured to be in contact with at least one adiposetissue and a second surface; the pressing and perforating unit furthercomprising at least one perforating member comprising a tip pointingtowards the first surface and configured to perforate at least oneadipose tissue by extending beyond the first surface, a leg attached tothe tip and a resilient connector connecting the tip to a strip; whereinthe pressing and perforating unit is configured to be placed on the baseof the bowl-like first pressing element.
 10. A method for preparing apreparation comprising a fat-depleted adipose tissue, the methodcomprising: mechanically removing a fat phase from at least one adiposetissue.
 11. (canceled)
 12. The method according to claim 10, wherein themechanically removing a fat phase from at least one adipose tissuecomprises: pressing the at least one adipose tissue.
 13. The methodaccording to claim 12, wherein the pressing at least one adipose tissuecomprises: pressing the at least one adipose tissue between a firstpressing element and a second pressing element.
 14. The method accordingto claim 10, further comprising: separating the fat phase from thefat-depleted adipose tissue.
 15. The method according to claim 10,further comprising: perforating the at least one adipose tissue.
 16. Themethod according to claim 10, further comprising: adding a preparationwhich is configured to enhance proliferation and differentiation ofmesenchymal stem cells.
 17. The method according to claim 10, furthercomprising: mixing with at least one type of bone tissue.
 18. The methodaccording to claim 10, further comprising: mixing with at least one typeof bone substitute.
 19. A preparation comprising a fat-depleted adiposetissue, wherein the fat-depleted adipose tissue is prepared bymechanically removing a fat phase from at least one adipose tissue. 20.(canceled)
 21. The preparation according to claim 19, further comprisingat least one type of bone tissue.
 22. The preparation according to claim19, further comprising at least one type of bone substitute.